Fig. 8

NGF facilitated the proliferation and metastasis of colon cancer through TrkA/ERK/ELK1/ZEB1 A. The expression of ERK1/2, phosphorylated ERK1/2, AKT, and phosphorylated AKT in SW480 cells upon administration of NGF were detected by Western blot and qRT-PCR. B. The expression of ELK1, phosphorylated ELK1, cMYC, and phosphorylated cMYC in SW480 cells upon administration of NGF were detected by Western blot and qRT-PCR. C. The schematic diagram exhibited one predicted binding site between ELK1 and the promoter of ZEB1. D. Western blot and qRT-PCR showed that knockdown of ELK1 decreased the expression of ZEB1 in SW480 and HCT116 cells. E. ChIP assay demonstrated the interaction between ELK1 and promoter of ZEB1 in SW480 and HCT116 cells. F. The vectors containing the wild-type (WT) or mutants (MUT) of ELK1 binding sites were co-transfected with or without si-ELK1 in colon cancer cells to perform the luciferase reporter assay. WT: wild type, MUT: Site1 mutated. G. ChIP assay demonstrated that administration of NGF significantly strengthened the accumulation of the ZEB1 promoter region combined with anti-ELK1 antibody, while knockdown of ELK1 reversed this increase. H. Luciferase reported assay showed that the enhanced activity of the ZEB1 promoter upon administration of NGF conspicuously blocked when knockdown of ELK1 in the WT group, whereas there was no significant alteration in the MUT group. WT: wild type, MUT: Site1 mutated. All data were revealed as means ± standard deviation (SD) for no less than three independent experiments. Significant P values showed as **P < 0.01, ***P < 0.001. n.s means the difference was not significant